CN108660129A - The method that sol-gal process fixes saccharase and the double enzymes of glucose oxidase - Google Patents
The method that sol-gal process fixes saccharase and the double enzymes of glucose oxidase Download PDFInfo
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Abstract
This application provides a kind of methods of fixed invertase and the double enzymes of glucose oxidase, include the following steps：(1) production of invertase；(2) invertase isolates and purifies；(3) after purification invertase and glucose oxidase solution is fixed on by sol-gal process on gel in the presence of silane coupling agent.Compared with traditional immobilization beta fructofuranosidase production lactosucrose, the present invention uses sol-gal process to fix invertase and the double enzymes of glucose oxidase to improve the stability of enzyme for the first time, obtain the higher enzyme activity rate of recovery, improve lactosucrose yield, it is easy to operate compared with conventional method, mild condition is a kind of environmentally friendly green method.
Description
Technical field
The present invention relates to technical field of enzyme engineering, logical more particularly, to a kind of saccharase and glucose oxidase
Cross the new method that sol-gal process realizes double enzyme co-immobilization production lactosucroses.
Background technology
Saccharase (EC 3.2.1.26) is a kind of glycosidase being widely present in plant and microorganism, both
With hydrolysis, energy catalysing sucrose is hydrolyzed to glucose and fructose, and has transglycosylation, can be widely used for complex functionality
Property oligosaccharide, food additives, the modification of glucosides substance and modification and other bioactive substance.But separate sources
Saccharase property is different, and ability and the receptor-specific for turning glycosyl are also different, and arthrobacterium produces saccharase
It is mainly used for catalyzing and synthesizing lactosucrose, can be transferred to the fructosyl that sucrose hydrolysis generates using sucrose and lactose as raw material
The C of lactose reducing end under neutral1On the hydroxyl of position, galactosyl sucrose, that is, lactosucrose is generated.
And since saccharase derives from microbial fermentation, there are the catalytic activity of a large amount of impurity, enzyme for enzyme preparation
It is low, stability is poor, the utilization rate of enzyme is low, resolvase is not easy to detach with product after reaction, reaction product purifying it is of high cost, it is difficult to
Realize industrialized production.And during synthesizing lactosucrose, saccharase also can catalysing sucrose and low
Poly- lactulose hydrolysis generates by-product glucose, and glucose meeting Noncompetition inhibition fructosidase turns fructosyl activity, makes oligomeric
Lactulose yield reduces, and glucose oxidase can be catalyzed glucose and be converted into gluconic acid, eliminates substrate and inhibits, improves oligomeric
Lactulose conversion ratio.
Enzyme immobilizatio is the new technology that the shortcomings that people are directed to resolvase grows up.It is fixed compared with free enzyme
Changing enzyme, there is lot of advantages, enzyme some structure changes may occur in immobilization process, to change it to a certain extent
Property and activity.And on the other hand, may make enzymatic structure rigidization to a certain extent by suitable method immobilized enzyme can prevent
Only enzyme denaturation, and the performances such as stability and activity are finally improved, realize the recycling of enzyme, and immobilised enzymes can also realize enzyme
With the separation of product, reduces product and isolate and purify cost.
Due to the fast development of oligosaccharide, the immobilization of saccharase is increasingly becoming the hot spot of research, but due to
Enzyme stability itself is poor, and the features such as being not easy to embed, about the fixed document of fructosidase and few, most commonly used research is shell
Glycan and sodium alginate investment, but these mechanical strengths are not high, activity of the immobilized enzyme retention rate is not high, exists substantially
60% or so, and activity declines obviously enzyme after a number of uses.
Sol-gal process is by the material mixings such as a certain amount of estersil, water, catalysts and solvents, shaking, by hydrolyzing, contracting
Close (including polycondensation), colloidal sol, aging, gel and cured method.Sol-gal process is with its mild reaction condition, extensive
Outstanding advantages of applicability and higher biomolecular structure and enzyme activity retention rate as immobilised enzymes effective ways.Due to
Silicon dioxide ratio organic polymer material is with more chemically and thermally stability, so applying relatively broad.But commonly molten
Sol-gel can make the activity release for the enzyme being embedded in gel be suppressed the fixation of saccharase, show
The enzyme activity gone out is substantially reduced, and can not more fix other enzymes simultaneously with saccharase so that its application receives certain
Limitation.
Invention content
In view of the above-mentioned problems existing in the prior art, the applicant provides a kind of saccharase and grape glycosyloxy
Change the method that enzyme realizes double enzyme co-immobilizations production lactosucroses by sol-gal process.With traditional immobilization beta-furans fruit
Glycosidase production lactosucrose compares, and the present invention uses sol-gal process to fix saccharase and glucose for the first time
The double enzymes of oxidizing ferment obtain the higher enzyme activity rate of recovery, lactosucrose yield are improved, with conventional method to improve the stability of enzyme
It is a kind of environmentally friendly green method compared to easy to operate, mild condition.
Technical scheme is as follows：
A kind of method of fixed saccharase and the double enzymes of glucose oxidase, includes the following steps：
(1) production of saccharase；
(2) saccharase isolates and purifies；
(3) after purification saccharase and glucose oxidase solution is in the presence of silane coupling agent by molten
Sol-gel is fixed on gel.
The method of the double enzymes of the fixation saccharase and glucose oxidase the specific steps are：
(1) production method of saccharase：
First Arthrobacter Arthrobacter sp.10137 are activated：Arthrobacterium is accessed in activation medium, in 30 DEG C
It is lower to cultivate for 24 hours, the strain activated；The activation medium is glucose 2%；Yeast extract 0.15%；Magnesium sulfate 0.01%；
Potassium dihydrogen phosphate 0.2%；And diammonium hydrogen phosphate 0.6%, the above unit are w/v；PH is 7.0-7.2；
Seed culture is carried out later：The strain after 1ml activation is drawn to the 250ml tapers that 100ml seed culture mediums are housed
In bottle；The seed culture medium is glucose 2%；Yeast extract 0.15%；Peptone 0.3%；Magnesium sulfate 0.01%；Biphosphate
Potassium 0.2%；And diammonium hydrogen phosphate 0.6%, the above unit are w/v, pH 7.0-7.2 are cultivated in shaking table at 30 DEG C
For 24 hours, it is 125r/min to shake speed；
Then fermented and cultured is carried out：Seed culture fluid is equipped with 100ml fermented and cultureds by 2%~4% inoculum concentration access
In the 250ml conical flasks of base, 24~48h is cultivated at 30 DEG C in shaking table, it is 125r/min to shake speed；The fermentation medium is sugarcane
Sugar 4%；Yeast extract 1.2%；Peptone 0.8%；Magnesium sulfate 0.2%；Potassium dihydrogen phosphate 0.2%；Diammonium hydrogen phosphate 0.4%, with
Upper unit is w/v；PH is 7.0-7.2；
The culture solution 4000r/min to have fermented is finally centrifuged into 20min, it is crude enzyme liquid to take out supernatant, cold at 4 DEG C
It hides spare；
(2) isolation and purification method of saccharase：
Using saturated ammonium sulfate salt fractionation method, ground solid sulfur is slowly added in the enzyme solution stirred first
Sour ammonium powder is sufficiently stirred 30~60min to 30% saturation degree, then puts 4 DEG C of refrigerators and stands 2~4h, take out supernatant, collects
Precipitation；
Continue to be slowly added to solid ammonium sulfate in the supernatant of taking-up to 40% saturation degree, takes out supernatant, it is heavy to collect
It forms sediment；
Ammonium sulfate is added to 50% saturation degree in continuation in the supernatant of taking-up, takes out supernatant, collects and precipitate, is more than repetition
Step adds ammonium sulfate to 60%, 70% saturation degree, finally to 80% saturation degree, takes out supernatant, collects precipitation, buffer solution is added
Dissolving precipitation；
The enzyme solution for gained of saltouing is added in bag filter, is dialysed in the environment of pH of buffer 6.5, dialyzate is changed every 8h
Once, purified enzyme solution is obtained；
(3) method that sol-gal process fixes saccharase and glucose oxidase after purification：
The 10ml PVA of 4%w/v are added in saccharase and glucose oxidase after purification under stirring
Solution, the 5ml NaF solution of a concentration of 1mol/L and 10ml water, stirring 30min~60min obtain homogeneous solution, then slowly add
Enter 3~5mL n-octytriethoxysilanes i.e. OTES and 6~20mL tetraethyl orthosilicates i.e. TEOS solution, continue to be stirred overnight,
Supernatant is poured out in centrifugation, is washed with water, and the gel for fixing double enzymes is obtained, and is added buffer solution to form coagulant liquid and is preserved.
Preferably, a concentration of 0.5~3.5mg/mL of saccharase described in step (3) after purification.
Preferably, the saccharase volume after purification being added described in step (3) is 10~40mL.
Preferably, a concentration of 0.5~2.5mg/mL of glucose oxidase described in step (3).
Preferably, the volume that glucose oxidase described in step (3) is added is 10~40mL.
Preferably, the volume ratio of the saccharase described in step (3) after purification and glucose oxidase is 1:3
~3:1.
Preferably, the volume ratio of OTES and TEOS described in step (3) is 1:2~1:4.
Preferably, arthrobacterium described in step (3) is purchased from Chinese industrial Organism Depositary, and deposit number is CICC
10137。
The applicant additionally provides the saccharase and the double enzymes of glucose oxidase fix after produce it is oligomeric
The method of lactulose：0.5ml substrate solutions are taken, the coagulant liquid that 0.5~4.5g fixes double enzymes is added, under conditions of pH6.5,
4~10h is reacted at 35~40 DEG C, is then centrifuged for, and supernatant, the solution after as reacting are taken out；The substrate solution includes
20% sucrose and 20% lactose, unit w/v.
The present invention is beneficial to be had technical effect that：
That the present invention overcomes common fixation methods is complicated for operation, of high cost, immobilization efficiency is not high, the enzyme activity rate of recovery is low,
The disadvantage that mechanical strength is low, operational stability is not high, the side of the saccharase provided and glucose oxidase immobilization
The method enzyme activity rate of recovery is high and stability is good.Initiator, n-octytriethoxysilane are mainly used as by sodium fluoride in the present invention
With tetraethyl orthosilicate as silane precursor, under liquid phase raw material is uniformly mixed using water as solvent, carry out a series of water
Solution, condensation reaction, form stable vitreosol system in the solution, and by ageing, micelle slowly polymerize, and constantly forms length
Chain forms gel network, and enzyme is wrapped in the inside, finally obtains and fixes the solidifying of saccharase and glucose oxidase
Glue.
The immobilised enzymes that the present invention prepares is convenient for recycling.Sol-gal process is fixed saccharase and Portugal
Grape carbohydrate oxidase provides new way, is the green method that a kind of step is simple, environmentally friendly.
The opposite enzyme activity of immobilized bi-enzyme prepared by the present invention compared with resolvase under acidic environment wants high more, fixation
Change double enzyme specific ionization enzymes more tolerant to acidic environment, there is wider range of pH value tolerance.In the environment of pH4.0, resolvase is protected
54.06% has been stayed, and immobilised enzymes remains with 85.38% enzyme activity.
The optimum temperature of immobilized bi-enzyme prepared by the present invention improves 5 DEG C compared with resolvase, and 40 are increased to from 35 DEG C
DEG C, when reaction temperature is increased to 55 DEG C, resolvase is almost without enzyme activity, and immobilized bi-enzyme still remains with 70.40% enzyme
It is living.
Immobilized bi-enzyme prepared by the present invention has good thermal stability compared with resolvase.Resolvase at 40 DEG C and with
It keeps the temperature 30min at upper temperature to lose activity, thermal stability is very poor, and half-life short, operational stability is poor, and cannot achieve repetition makes
With.And the optimal reactive temperature of immobilised enzymes is 40 DEG C, and the enzyme activity that 30min still remains with 51.85% is kept the temperature at 60 DEG C.
Immobilised enzymes recycling step prepared by the present invention is simple, and supernatant is taken out in centrifugation after reaction, is added new
Reaction solution the recycling of enzyme can be realized in precipitation；The enzyme activity rate of recovery is high, and the enzyme activity rate of recovery reaches 85.39%；And again
Multiple stability in use is good, and the enzyme activity after reusing 8 times retains up to 82%.
This method is easy to operate, of low cost, and the immobilized bi-enzyme of preparation is in continuous production lactosucrose and raising
The fields such as lactosucrose yield have wide application prospect.
Description of the drawings
Fig. 1 is the process flow chart that sol-gal process fixes saccharase and the double enzymes of glucose oxidase.
Fig. 2 is the reusability of immobilised enzymes prepared by embodiment 3.
Specific implementation mode
1 method indicated and specific embodiment below in conjunction with the accompanying drawings, are specifically described the present invention.
Embodiment 1：
First by Arthrobacter sp.10137 actication of culture, the arthrobacterium of preservation is accessed into activation medium (glucose
2% (w/v)；Yeast extract 0.15% (w/v)；Magnesium sulfate 0.01% (w/v)；Potassium dihydrogen phosphate 0.2% (w/v)；Diammonium hydrogen phosphate
0.6% (w/v)；PH7.0-7.2 in), the strain activated for 24 hours is cultivated at 30 DEG C.Seed culture is carried out later, is drawn
Strain after 1ml activation is to equipped with 100ml seed culture mediums (glucose 2% (w/v)；Yeast extract 0.15% (w/v)；Peptone
0.3% (w/v)；Magnesium sulfate 0.01% (w/v)；Potassium dihydrogen phosphate 0.2% (w/v)；Diammonium hydrogen phosphate 0.6% (w/v), pH7.0-
7.2) it in 250ml conical flasks, is cultivated for 24 hours at 30 DEG C in shaking table, it is 125r/min to shake speed.Then fermented and cultured is carried out, it will
Seed culture fluid is equipped with 100ml fermentation mediums (sucrose 4% (w/v) by 2% inoculum concentration access；Yeast extract 1.2% (w/v)：
Peptone 0.8% (w/v)；Magnesium sulfate 0.2% (w/v)；Potassium dihydrogen phosphate 0.2% (w/v)；Diammonium hydrogen phosphate 0.4% (w/v),
PH7.0-7.2 in 250ml conical flasks), 48h is cultivated at 30 DEG C in shaking table, it is 125r/min to shake speed.It will finally ferment
Culture solution 4000r/min centrifuges 20min, and it is crude enzyme liquid to take out supernatant, is refrigerated at 4 DEG C spare.
Using saturated ammonium sulfate salt fractionation method, ground solid sulfur is slowly added in the enzyme solution stirred first
Sour ammonium powder is sufficiently stirred 30min to 30% saturation degree, then puts 4 DEG C of refrigerators and stands 2h, take out supernatant, collects precipitation.
Continue to be slowly added to solid ammonium sulfate in the supernatant of taking-up to 40% saturation degree, takes out supernatant, collect precipitation.Continuation is taking
Ammonium sulfate is added in the supernatant gone out to 50% saturation degree, takes out supernatant, collects precipitation, repeat above step, add ammonium sulfate extremely
60%, 70% saturation degree takes out supernatant finally to 80% saturation degree, collects precipitation, and buffer solution precipitation is added.It will saltout
The enzyme solution of gained is added in bag filter, dialyses in the environment of pH of buffer 6.5, dialyzate changes once every 8h, is purified
Good enzyme solution.
Saccharase 10mL (1mg/mL) and glucose oxidase 20mL (1mg/mL, pH5.0) after purification is stirred
It mixes and 10ml PVA solutions (4%, w/v), 5ml NaF solution (1mol/L) and 10ml water is added under state, stirring 30min is obtained uniformly
Solution is then slowly added into 4mL n-octytriethoxysilanes (OTES) and 10mL tetraethyl orthosilicates (TEOS) solution, continues
It is stirred overnight, supernatant is poured out in centrifugation, is washed with water 3 times.
Embodiment 2：
First by Arthrobacter sp.10137 actication of culture, the arthrobacterium of preservation is accessed into activation medium (glucose
2% (w/v)；Yeast extract 0.15% (w/v)；Magnesium sulfate 0.01% (w/v)；Potassium dihydrogen phosphate 0.2% (w/v)；Diammonium hydrogen phosphate
0.6% (w/v)；PH7.0-7.2 in), the strain activated for 24 hours is cultivated at 30 DEG C.Seed culture is carried out later, is drawn
Strain after 1ml activation is to equipped with 100ml seed culture mediums (glucose 2% (w/v)；Yeast extract 0.15% (w/v)；Peptone
0.3% (w/v)；Magnesium sulfate 0.01% (w/v)；Potassium dihydrogen phosphate 0.2% (w/v)；Diammonium hydrogen phosphate 0.6% (w/v), pH7.0-
7.2) it in 250ml conical flasks, is cultivated for 24 hours at 30 DEG C in shaking table, it is 125r/min to shake speed.Then fermented and cultured is carried out, it will
Seed culture fluid is equipped with 100ml fermentation mediums (sucrose 4% (w/v) by 3% inoculum concentration access；Yeast extract 1.2% (w/v)：
Peptone 0.8% (w/v)；Magnesium sulfate 0.2% (w/v)；Potassium dihydrogen phosphate 0.2% (w/v)；Diammonium hydrogen phosphate 0.4% (w/v),
PH7.0-7.2 in 250ml conical flasks), 36h is cultivated at 30 DEG C in shaking table, it is 125r/min to shake speed.It will finally ferment
Culture solution 4000r/min centrifuges 20min, and it is crude enzyme liquid to take out supernatant, is refrigerated at 4 DEG C spare.
Using saturated ammonium sulfate salt fractionation method, ground solid sulfur is slowly added in the enzyme solution stirred first
Sour ammonium powder is sufficiently stirred 60min to 30% saturation degree, then puts 4 DEG C of refrigerators and stands 3h, take out supernatant, collects precipitation.
Continue to be slowly added to solid ammonium sulfate in the supernatant of taking-up to 40% saturation degree, takes out supernatant, collect precipitation.Continuation is taking
Ammonium sulfate is added in the supernatant gone out to 50% saturation degree, takes out supernatant, collects precipitation, repeat above step, add ammonium sulfate extremely
60%, 70% saturation degree takes out supernatant finally to 80% saturation degree, collects precipitation, and buffer solution precipitation is added.It will saltout
The enzyme solution of gained is added in bag filter, dialyses in the environment of pH of buffer 6.5, dialyzate changes once every 8h, is purified
Good enzyme solution.
Saccharase 30mL (2.5mg/mL) after purification and glucose oxidase 15mL (2mg/mL, pH5.0)
10ml PVA solutions (4%, w/v), 5ml NaF solution (1mol/L) and 10ml water are added under stirring, stirring 60min is obtained
Even solution is then slowly added into 5mL n-octytriethoxysilanes (OTES) and 15mL tetraethyl orthosilicates (TEOS) solution, after
Continuous to be stirred overnight, supernatant is poured out in centrifugation, is washed with water 3 times.
Embodiment 3：
First by Arthrobacter sp.10137 actication of culture, the arthrobacterium of preservation is accessed into activation medium (glucose
2% (w/v)；Yeast extract 0.15% (w/v)；Magnesium sulfate 0.01% (w/v)；Potassium dihydrogen phosphate 0.2% (w/v)；Diammonium hydrogen phosphate
0.6% (w/v)；PH7.0-7.2 in), the strain activated for 24 hours is cultivated at 30 DEG C.Seed culture is carried out later, is drawn
Strain after 1ml activation is to equipped with 100ml seed culture mediums (glucose 2% (w/v)；Yeast extract 0.15% (w/v)；Peptone
0.3% (w/v)；Magnesium sulfate 0.01% (w/v)；Potassium dihydrogen phosphate 0.2% (w/v)；Diammonium hydrogen phosphate 0.6% (w/v), pH7.0-
7.2) it in 250ml conical flasks, is cultivated for 24 hours at 30 DEG C in shaking table, it is 125r/min to shake speed.Then fermented and cultured is carried out, it will
Seed culture fluid is equipped with 100ml fermentation mediums (sucrose 4% (w/v) by 4% inoculum concentration access；Yeast extract 1.2% (w/v)：
Peptone 0.8% (w/v)；Magnesium sulfate 0.2% (w/v)；Potassium dihydrogen phosphate 0.2% (w/v)；Diammonium hydrogen phosphate 0.4% (w/v),
PH7.0-7.2 in 250ml conical flasks), 48h is cultivated at 30 DEG C in shaking table, it is 125r/min to shake speed.It will finally ferment
Culture solution 4000r/min centrifuges 20min, and it is crude enzyme liquid to take out supernatant, is refrigerated at 4 DEG C spare.
Using saturated ammonium sulfate salt fractionation method, ground solid sulfur is slowly added in the enzyme solution stirred first
Sour ammonium powder is sufficiently stirred 60min to 30% saturation degree, then puts 4 DEG C of refrigerators and stands 4h, take out supernatant, collects precipitation.
Continue to be slowly added to solid ammonium sulfate in the supernatant of taking-up to 40% saturation degree, takes out supernatant, collect precipitation.Continuation is taking
Ammonium sulfate is added in the supernatant gone out to 50% saturation degree, takes out supernatant, collects precipitation, repeat above step, add ammonium sulfate extremely
60%, 70% saturation degree takes out supernatant finally to 80% saturation degree, collects precipitation, and buffer solution precipitation is added.It will saltout
The enzyme solution of gained is added in bag filter, dialyses in the environment of pH of buffer 6.5, dialyzate changes once every 8h, is purified
Good enzyme solution.
Saccharase 40mL (2mg/mL) after purification and glucose oxidase 40mL (1.5mg/mL, pH5.0)
10ml PVA solutions (4%, w/v), 5ml NaF solution (1mol/L) and 10ml water are added under stirring, stirring 30min is obtained
Even solution is then slowly added into 3mL n-octytriethoxysilanes (OTES) and 8mL tetraethyl orthosilicates (TEOS) solution, after
Continuous to be stirred overnight, supernatant is poured out in centrifugation, is washed with water 3 times.
Embodiment 4
0.5ml substrate solutions (20% (w/v) sucrose+20% (w/v) lactose) are taken, are added embodiment 3 is prepared 0.5
~4.5ml fixes the coagulant liquid of double enzymes, and under conditions of pH6.5,8h is reacted at 35 DEG C, and 10000r/min centrifuges 10min,
Supernatant is taken out, the solution after as reacting detects lactosucrose content.Immobilization beta-furans of 0.5~4.5ml is added
Fructosidase list enzyme is control, calculates lactosucrose yield.
0.5ml substrate solutions (20% (w/v) sucrose+20% (w/v) lactose) are taken, are added embodiment 3 is prepared 0.5
~4.5ml fixes the coagulant liquid of double enzymes, and under conditions of pH6.5,8h is reacted at 35 DEG C, and 10000r/min centrifuges 10min,
Supernatant is taken out, the solution after as reacting detects lactosucrose content, and substrate solution is continuously added in precipitation and is carried out instead
It answers, then discards reaction solution again, repeat reaction 8 batches, realize the reuse of immobilized bi-enzyme, obtain Fig. 2, immobilized bi-enzyme
Reusability.
The results show that compared with immobilization saccharase list enzyme, it is added oligomeric in the reaction solution of immobilized bi-enzyme
Lactulose yield improves 15%.And immobilized bi-enzyme has good reusability, after using 8 times, also retains
82% enzyme activity, operational stability are greatly improved.The enzyme molecular structure of immobilized bi-enzyme is relatively stablized, therefore has
Good stability can be repeated as many times and use, and high mechanical strength, and the reaction column to do continuous production below is established
Basis substantially increases economic benefit and the yield of lactosucrose.
Claims (10)
1. a kind of method of fixed saccharase and the double enzymes of glucose oxidase, it is characterised in that include the following steps：
(1) production of saccharase；
(2) saccharase isolates and purifies；
(3) after purification saccharase and glucose oxidase solution is solidifying by colloidal sol in the presence of silane coupling agent
Glue method is fixed on gel.
2. the method for fixed saccharase according to claim 1 and the double enzymes of glucose oxidase, feature exist
In the specific steps are：
(1) production method of saccharase：
First Arthrobacter Arthrobacter sp.10137 are activated：Arthrobacterium is accessed in activation medium, is trained at 30 DEG C
It supports for 24 hours, the strain activated；The activation medium is glucose 2%；Yeast extract 0.15%；Magnesium sulfate 0.01%；Phosphoric acid
Potassium dihydrogen 0.2%；And diammonium hydrogen phosphate 0.6%, the above unit are w/v；PH is 7.0-7.2；
Seed culture is carried out later：It draws in the strain to the 250ml conical flasks equipped with 100ml seed culture mediums after 1ml activation；
The seed culture medium is glucose 2%；Yeast extract 0.15%；Peptone 0.3%；Magnesium sulfate 0.01%；Potassium dihydrogen phosphate
0.2%；And diammonium hydrogen phosphate 0.6%, the above unit are w/v, pH 7.0-7.2 are cultivated for 24 hours in shaking table at 30 DEG C,
It is 125r/min to shake speed；
Then fermented and cultured is carried out：Seed culture fluid is equipped with 100ml fermentation mediums by 2%~4% inoculum concentration access
In 250ml conical flasks, 24~48h is cultivated at 30 DEG C in shaking table, it is 125r/min to shake speed；The fermentation medium is sucrose
4%；Yeast extract 1.2%；Peptone 0.8%；Magnesium sulfate 0.2%；Potassium dihydrogen phosphate 0.2%；Diammonium hydrogen phosphate 0.4%, it is above
Unit is w/v；PH is 7.0-7.2；
The culture solution 4000r/min to have fermented is finally centrifuged into 20min, it is crude enzyme liquid to take out supernatant, is refrigerated at 4 DEG C standby
With；
(2) isolation and purification method of saccharase：
Using saturated ammonium sulfate salt fractionation method, ground solid ammonium sulfate is slowly added in the enzyme solution stirred first
Powder is sufficiently stirred 30~60min to 30% saturation degree, then puts 4 DEG C of refrigerators and stands 2~4h, takes out supernatant, and it is heavy to collect
It forms sediment；
Continue to be slowly added to solid ammonium sulfate in the supernatant of taking-up to 40% saturation degree, takes out supernatant, collect precipitation；
Continue to be added ammonium sulfate in the supernatant of taking-up to 50% saturation degree, takes out supernatant, collect precipitation, repeat the above step
Suddenly, add ammonium sulfate to 60%, 70% saturation degree, finally to 80% saturation degree, take out supernatant, collect precipitation, it is molten that buffer solution is added
Solution precipitation；
The enzyme solution for gained of saltouing is added in bag filter, is dialysed in the environment of pH of buffer 6.5, dialyzate changes one every 8h
It is secondary, obtain purified enzyme solution；
(3) method that sol-gal process fixes saccharase and glucose oxidase after purification：
Saccharase and glucose oxidase after purification be added under stirring 4%w/v 10ml PVA solutions,
The 5ml NaF solution and 10ml water of a concentration of 1mol/L, stirring 30min~60min obtain homogeneous solution, it is then slowly added into 3~
5mL n-octytriethoxysilanes, that is, OTES and 6~20mL tetraethyl orthosilicates, that is, TEOS solution, continue to be stirred overnight, centrifugation
Supernatant is poured out, is washed with water, the gel for fixing double enzymes is obtained, adds buffer solution to form coagulant liquid and is preserved.
3. the method for fixed saccharase according to claim 2 and the double enzymes of glucose oxidase, feature exist
A concentration of 0.5~3.5mg/mL of saccharase described in step (3) after purification.
4. the method for fixed saccharase according to claim 2 and the double enzymes of glucose oxidase, feature exist
The saccharase volume after purification being added described in step (3) is 10~40mL.
5. the method for fixed saccharase according to claim 2 and the double enzymes of glucose oxidase, feature exist
A concentration of 0.5~2.5mg/mL of glucose oxidase described in step (3).
6. the method for fixed saccharase according to claim 2 and the double enzymes of glucose oxidase, feature exist
The volume that the glucose oxidase described in step (3) is added is 10~40mL.
7. the method for fixed saccharase according to claim 2 and the double enzymes of glucose oxidase, feature exist
The volume ratio of saccharase and glucose oxidase described in step (3) after purification is 1:3~3:1.
8. the method for fixed saccharase according to claim 2 and the double enzymes of glucose oxidase, feature exist
The volume ratio of OTES and TEOS described in step (3) is 1:2~1:4.
9. the method for fixed saccharase according to claim 2 and the double enzymes of glucose oxidase, feature exist
The arthrobacterium described in step (3) is purchased from Chinese industrial Organism Depositary, and deposit number is CICC 10137.
10. claim 1~9 any one of them saccharase and the double enzymes of glucose oxidase produce oligomeric after fixing
The method of lactulose, it is characterised in that：0.5ml substrate solutions are taken, the coagulant liquid that 0.5~4.5g fixes double enzymes is added,
Under conditions of pH6.5,4~10h is reacted at 35~40 DEG C, is then centrifuged for, take out supernatant, the solution after as reacting；Institute
It includes 20% sucrose and 20% lactose, unit w/v to state substrate solution.
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